Since T₁/R₁ changes induced by different Mn²⁺ concentrations (i.e. R₁=R₁ (0)+γ1×[Mn²⁺]) were generally measured in solution and was assumed very similar to in vivo conditions. We determined the effects of different manganese concentrations on T₁/R₁ in solution with the following concentrations up to 1000µmol/L.

All measurements were performed at 14T. Gradient echo (GRE) MRI was applied for anatomical images and with varying inversion times(TIs) to obtain T₁/R₁. Inversion-recovery-spin-echo (IR-SE) MRI was adjusted to null pancreatic tissue (based on T₁) and to monitor a manganese uptake-induced signal enhancement. Inversion-recovery look-locker GRE MRI with three TRs (5/10/20sec) was used to measure T₁* (R₁*). Region of interest (ROI) was drawn manually based on the anatomical images.

Adult male C57BL/6J mice (~25g) were used. Diabetes was induced by streptozotocin (STZ, 180mg/kg) in mice (~35g). One week after the STZ treatments, all diabetic mice were weighing ~25g and measured. All animals were anesthetized under isoflurane (1-2%) mixed with air and oxygen (1:1), and were continuously monitored for their breathing rates (80-100bpm) and body temperature (35-37°C) through a MR-compatible monitor system, which can also deliver desired triggering signals (TTL) for respiration-gating all MRI acquisitions. All mice were warmed through a heating pad, which did not interfere with the function of a volume coil (50-mm-diameter, 32-mm-long) used for this study (2). Three conditions were compared: normoglycemia (fasting, without glucose, a manganese (a 15mM MnCl₂ solution) infusion, i.e. 0.2ml/hr/25g), hyperglycemia (150µl 20% glucose plus injected 5-min prior to the manganese infusion); and STZ-induced diabetes (with a bolus of 150µl 20% glucose, injected 5-min prior to the manganese infusion).

With the respiration gating, MR images were acquired with minimal motion artifacts and satisfactory quality. Such images allowed measuring T₁ of pancreas and other surrounding tissues of living mouse at 14.1T, as shown in Figure 1. The R₁ changes induced by manganese concentrations in solution at 14.1T was linearly correlated, γ₁= (82±0.2) [s^-1(mmol/L)^-1] (R²= 0.99, p<0.0001, Figure 2). The apparent T₁ (R₁*) from the Look-locker GRE approach (FOV=35×32mm2, RO×PE=128X96, TE/TR=3/5000ms) was consistently 1.29±0.04 times higher but highly correlated (R= 0.99, p<0.0001) that R₁ from the standard IR-SE acquisitions (Fig.2).

The signal originated from the pancreas without MnCl₂ was nearly nulled at TI=700ms, which was based on T1 of pancreas at 14T. Upon i.v. infusion of MnCl₂, the signal became above the noise levels and more apparent from the 10 min post-injection and attended its higher levels at 30 min post-injection (Fig. 3), which imitated a pseudo-first order association kinetics of the interaction to the manganese uptakes via the calcium channels. The signal persisted even 30-min after the end of the infusion, as the limits of the gland were easy to delimitate from the surrounding non-pancreatic tissues (Fig.3). Once the MEMRI signal was normalized on the signal obtained before Mn²⁺ infusion, the amplitude of the MEMRI signal after manganese infusion was lowest in STZ-induced diabetic mice; significantly increased in normoglycemic mice; and further elevated in hyperglycemic mice (Fig.4). The signal changes induced by manganese uptakes in both pancreas and kidney were significantly different from each other among three groups (p<0.0001, Fig. 3&4).

Further measurements of R₁* shortly after the infusion protocol showed that the R₁* of the STZ-treated pancreata was 1.49 ± 0.14s^-1, higher than 0.94 ± 0.09s-1 of healthy controls without either glucose or MnCl₂ (p=0.004, Fig 4B), but 50.4% less than 3.00 ± 0.36s^-1 of healthy mice with both glucose and MnCl₂ (p<0.001, Fig. 4B). In contrast, the decrease of volume density (Vvi) of the islets upon STZ treatment, determined by morphometry, was of 51.8% (Fig 3B, ref 1). The manganese contents in pancreas of these three groups were then estimated based on the R₁* changes to be 52±2µmol/L in STZ, 90±3μmol/L in control without glucose and 208±4µmol/L in healthy mice with glucose (Fig. 4).

Given the non-specific uptake of manganese by various cell types and the well-maintained acini upon STZ treatments, our results suggest that the MEMRI signals under the three conditions might reflect different pancreatic b-cell functions through the islet-acini axis. We concluded that MEMRI provided quantitative evidence of manganese uptake in pancreas of living mice.